Parallel high-performance liquid chromatography with post-separation treatment

ABSTRACT

High-performance liquid chromatography (HPLC) methods and systems are disclosed that combine parallel chromatographic separation of a plurality of samples with a detection technique that involves post-separation treatment of the plurality of samples to enhance one or more properties of the sample or of a component thereof, followed by detection of the one or more enhanced properties. Selective, tunable detection schemes are achievable, and are particularly advantageous as applied in connection with combinatorial chemistry, combinatorial material science and more particularly, combinatorial synthesis and screening of polymeric materials.

[0001] This application claims priority to the following U.S. patentapplications, each of which is hereby incorporated by reference for allpurposes: Ser. No. 09/285,363 entitled “Rapic Characterization ofPolymers”, filed Apr. 2, 1999 by Petro et al.; Ser. No. 09/285,393entitled “Automated Sampling Methods for Rapid Characterization ofPolymers”, filed Apr. 2, 1999 by Petro et al.; Ser. No. 09/285,333entitled “High-Temperature Characterization of Polymers”, filed Apr. 2,1999 by Petro et al.; Ser. No. 09/285,335 entitled “Flow-InjectionAnalysis and Variable-Flow Light Scattering Apparatus and Methods forCharacterizing Polymers”, filed Apr. 2, 1999 by Nielsen et al.; and Ser.No. 09/285,392 entitled “Indirect Calibration of PolymerCharacterization Systems”, filed Apr. 2, 1999 by Petro et al. Thisapplication is related to U.S. patent application Ser. No. 09/410,546entitled “Parallel High-Performance Liquid Chromatography With SerialInjection” filed by Petro et al. on Oct. 1, 1999, under Attorney DocketNo. 99-79, and is hereby incorporated by reference for all purposes.

BACKGROUND OF INVENTION

[0002] The present invention generally relates to liquid chromatography,and specifically, to high-pressure liquid chromatography (HPLC) methodsfor rapidly characterizing a plurality of samples. The inventionparticularly relates, in a preferred embodiment, to parallel HPLCmethods for characterizing a combinatorial library comprising differentpolymers.

[0003] Liquid chromatography is generally well known in the art.High-pressure liquid chromatographic techniques involve injection of asample into a mobile-phase that flows through a chromatographic column,separation of one or more components of the sample from other componentsthereof in the chromatographic column, and detection of the separatedcomponents with a flow-through detector. Approaches for liquidchromatography typically vary, however, with respect to the basis ofseparation and with respect to the basis of detection.

[0004] Gel permeation chromatography (GPC), a well-known form of sizeexclusion chromatography (SEC), is a frequently-employed chromatographictechnique for separation of samples generally, and for polymer sizedetermination particularly. Another chromatographic separation approachis illustrated by U.S. Pat. 5,334,310 to Frechet et al. and involves theuse of a porous monolithic stationary-phase as a separation mediumwithin the chromatographic column, combined with a mobile-phasecomposition gradient. Other separation approaches are also known in theart, including for example, normal-phase adsorption chromatography, andreverse-phase chromatography.

[0005] After separation, a detector can measure a property of the sampleor of a sample component—from which one or more characterizingproperties, such as molecular weight can be determined as a function oftime. Specifically, with respect to polymer samples, for example, anumber of molecular-weight related parameters can be determined,including for example: the weight-average molecular weight (M_(w)), thenumber-average molecular weight (M_(n)), the molecular-weightdistribution shape, and an index of the breadth of the molecular-weightdistribution (M_(w)/M_(n)), known as the polydispersity index (PDI).Other characterizing properties, such as mass, particle size,composition or conversion can likewise be determined. A variety ofcontinuous-flow detectors have been used for measurements in liquidchromatography systems. Common flow-through detectors include opticaldetectors such as a differential refractive index detector (RI), anultraviolet-visible absorbance detector (UV-VIS), or an evaporative massdetector (EMD) sometimes referred to as an evaporative light scatteringdetector (ELSD). Additional detection instruments, such as astatic-light-scattering detector (SLS), a dynamic-light-scatteringdetector (DLS), and/or a capillary-viscometric detector (CNV) arelikewise known for measurement of properties of interest.

[0006] Detection methods involving post-separation treatment are knownin the art. With respect to polymer samples, for example, EuropeanPatent EP 675 356 B1 to Staal discloses a method and a system forprecipitating polymer components of a sample in the effluent stream froma chromatographic column, with optical detection of the precipitatedcomponents, and is hereby incorporated by reference for all purposes.Significantly, the application of such detection methods to parallelHPLC systems was not contemplated, and the benefits thereof were notheretofore appreciated in the art.

[0007] Broadly available liquid chromatography systems are not entirelysatisfactory for efficiently screening larger numbers of samples. Withrespect to polymers, for example, high-performance liquidchromatographic techniques can typically take up to an hour for eachsample to ensure a high degree of separation over the wide range ofpossible molecular weights (i.e., hydrodynamic volumes) for a sample.Notably, however, substantial improvements in sample throughput havebeen achieved in the art. For example, rapid-serial approaches forcharacterizing polymers have been developed by Symyx Technologies, Inc.(Santa Clara, Calif.) and disclosed in the aforementioned co-pendingU.S. patent applications from which the present application claimspriority. As another example, U.S. Pat. No. 5,783,450 to Yoshida et al.discloses rapid-serial protocols and systems for preparation,purification and separation of small molecules such as catecholaminesand protaglandins from biological samples such as blood.

[0008] Parallel approaches for liquid chromatography have also beencontemplated in the art. Zeng et al., Development of a Fully AutomatedParallel HPLC/Mass Spectrometry System for the AnalyticalCharacterization and Preparative Purification of CombinatorialLibraries, Anal. Chem. 70, 4380-4388 (1998), disclose analytical andpreparative HPLC methods and systems involving the sequential preloadingof samples onto two chromatographic columns, and then applying amobile-phase in parallel to each of the columns to effect parallelseparation of the samples. According to an alternative approachdisclosed in U.S. Pat. No. 5,766,481 to Zambias et al., parallelseparation of a plurality of molecules is effected by forming a mixtureof selected, compatible molecules, and subsequently resolving themixture sample into its component molecules by separation in asingle-channel HPLC system. Parallel approaches have likewise beenemployed in other separation protocols, such as capillaryelectrophoresis. See, for example, U.S. Pat. No. 5,900,934 to Gilby etal.

[0009] Although such parallel approaches and systems have been generallycontemplated, there nonetheless exists a need in the art for improvingsuch approaches and systems with respect to overall sample throughputand/or quality of data. Moreover, with the development of combinatorialmaterials science techniques that allow for the parallel synthesis oflibraries comprising a vast number of diverse industrially relevantmaterials, and especially polymeric materials, there is a need for HPLCmethods and systems to rapidly characterize the properties of samplesfrom such combinatorial libraries.

SUMMARY OF INVENTION

[0010] It is therefore an object of the present invention to provideHPLC systems and protocols having a higher overall sample throughput,and in preferred applications, employing such systems and protocols forcharacterizing combinatorial libraries of material samples such aspolymer samples, and particularly, libraries of or derived frompolymerization product mixtures, to facilitate the discovery ofcommercially important materials such as polymeric materials, catalysts,polymerization conditions and/or post-synthesis processing conditions.

[0011] Briefly, therefore, the present invention is directed to methodsand systems for characterizing a plurality of samples by liquidchromatography or, in some embodiments, by flow-injection analysis.According to the methods, a mobile phase is supplied in parallel througheach of first and second chromatographic columns of a high-pressureliquid chromatography system. First and second samples are injected intothe mobile phase of the first and second chromatographic columns,respectively. In chromatographic applications, at least one samplecomponent of the injected first and second samples is separated fromother sample components thereof in the respective chromatographiccolumns. Significantly, after separation, but before detection, at leastone separated sample component of the first and second samples istreated to change a property of at least one separated sample componentthereof. The treatment is preferably precipitation and/orderivitization. A property of the treated sample component of the firstand second samples is detected.

[0012] The present invention provides substantial advantages over knownapproaches for parallel liquid chromatography systems. High overallthroughput is achieved with a parallel HPLC system—even for samples(e.g., many types of polymers) having components that would otherwise bedifficult to detect, and moreover, in a cost-effective manner. Theseadvantages are particularly realized in preferred embodiments, in whichoptical detectors are employed. Moreover, methods of the invention canbe advantageously employed in connection with mini- and micro-scaleparallel liquid chromatography systems, since optical detectors arereadily miniaturized—even to macro-scale applications.

[0013] Other features, objects and advantages of the present inventionwill be in part apparent to those skilled in art and in part pointed outhereinafter. All references cited in the instant specification areincorporated by reference for all purposes. Moreover, as the patent andnon-patent literature relating to the subject matter disclosed and/orclaimed herein is substantial, many relevant references are available toa skilled artisan that will provide further instruction with respect tosuch subject matter.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1A through FIG. 1F are schematic diagrams showing an overviewof polymer characterization process steps (FIG. 1A), a rapid-serialprotocol for effecting such steps (FIG. 1B) for a plurality of samples(s₁, s₂, s₃ . . . s_(n)) to obtain corresponding characterizing propertyinformation (p₁, p₂, p₃ . . . p_(n)), a parallel protocol for effectingsuch steps (FIG. 1C) and several parallel-serial hybrid protocols foreffecting such steps (FIG. 1D, FIG. 1E, FIG. 1F).

[0015]FIG. 2A through FIG. 2C are schematic diagrams illustratingembodiments for the application of post-separation treatment to parallelHPLC systems having four chromatographic channels and four dedicatedparallel detectors (FIG. 2A and FIG. 2B) or a single detector with adetection switching valve (FIG. 2C).

[0016]FIG. 3A and FIG. 3B are schematic diagrams illustrating variousparallel high-performance liquid chromatography systems as embodimentsof the invention having four chromatographic channels with fourdedicated sample loading ports and injection valves (FIG. 3A) or asingle sample loading port and single injection valve with a multiportswitching valve (FIG. 3B).

[0017]FIG. 4 is a schematic diagram illustrating an eight-port injectionvalve that can be used (e.g.,in connection with a multi-port switchingvalve) for loading a polymer sample and for injection thereof into amobile phase of a flow characterization system.

[0018]FIG. 5 is a schematic diagram illustrating an automated samplingsystem.

[0019]FIG. 6 is a graph of detector output (mv, absorbance at 350 nm)versus time (minutes) illustrating the results from a HPLC separation ofnarrow polydispersity polyisobutylene standard having a molecular weightof 1M in a tetrahydrofuran mobile phase/eluant, and run with variouspost-separation precipitation protocols: (i) without treatment of thechromatographic column effluent (unmodified control); (ii) withtreatment of the chromatographic column effluent with methanol at a flowrate of 1 ml/min; and (iii) with treatment of the chromatographic columneffluent with water at a flow rate of 0.2 ml/min (See Example 1).

[0020]FIG. 7A and FIG. 7B are graphs of detector output (mv, absorbanceat 350 nm) versus time (minutes) illustrating the results from a HPLCseparation of narrow polydispersity polyisobutylene (PIB) standards(FIG. 7A) and narrow polydispersity polystyrene (PS) standards (FIG. 7B)of various molecular weights (PIB at 1M, 355 k and 10K; and PS at 3M,215 k and 11 k) each in a tetrahydrofuran mobile phase/eluant, run: (i)without treatment of the chromatographic column effluent (unmodifiedcontrol) and (ii)with treatment of the chromatographic column effluentwith water at a flow rate of 0.1 ml/min (See Example 2).

[0021]FIG. 8A through FIG. 8D are graphs of detector output (mv,absorbance at 350 nm) versus time (minutes) illustrating the resultsfrom a HPLC separation of narrow polydispersity polystyrene (PS)standards of various molecular weights (3M, 70 k and 11 k) each in atetrahydrofuran mobile phase/eluant, run with various treatments of thechromatographic column effluent: with water at a flow rate of 0.5 ml/min(FIG. 8A); with water at a flow rate of 0.3 ml/min (FIG. 8B); with waterat a flow rate of 0.15 ml/min (FIG. 8C); and with water at a flow rateof 0.1 ml/min (FIG. 8D) (See Example 3).

[0022]FIG. 9A and FIG. 9B are graphs of detector output (mv) versus time(minutes) with detector output be based on UV absorbance at 254 nm (FIG.9A) or static light scattering (SLS) at 90° (FIG. 9B), illustrating theresults from a HPLC separation of narrow polydispersity polystyrene (PS)standard having a molecular weight of 214 k in a tetrahydrofuran mobilephase/eluant, run: (i) without treatment of the chromatographic columneffluent (unmodified control) and (ii) with treatment of thechromatographic column effluent with water at a flow rate of 0.4 ml/min(See Example 4). The detector responses are in the same scale for bothoverlaid traces of each figure.

DETAILED DESCRIPTION OF THE INVENTION

[0023] In the present invention, methods and apparatus having featuresthat enable an effective combinatorial materials research program areprovided. Such a research program may be directed, for example, toidentifying or optimizing commercially valuable polymers, catalysts orother materials, or to other research goals, such as processcharacterization and optimization. Other applications, includingparallel industrial process monitoring or control are also enabled bythe present invention.

[0024] Sample characterization approaches and systems of the inventioninvolve parallel HPLC approaches and systems combined withpost-separation treatment protocols and systems. Specifically, aplurality of samples are separated in parallel in two or morechromatographic columns, and the samples and/or one or more separatedcomponents thereof are subsequently treated, for example with aprecipitating or derivatizing agent, to change one or more properties ofthe one or more components—for enhanced and/or selectively enhanceddetection of such property or properties. In preferred approaches, thetreatment effects a change in an optical property, and the detectedproperty of the treated sample and/or component is an optical property.

[0025] The present invention is preferably applied to, and primarilydiscussed in connection with, combinatorial chemistry, combinatorialmaterial science and more particularly, combinatorial synthesis andscreening of polymeric materials. Briefly, in a combinatorial approachfor identifying or optimizing materials (e.g., polymers) or reactionconditions, a large compositional space (e.g., of monomers, comonomers,catalysts, catalyst precursors, solvents, initiators, additives, or ofrelative ratios of two or more of the aforementioned) and/or a largereaction condition space (e.g., of temperature, pressure and reactiontime) may be rapidly explored by preparing libraries of diversematerials and then rapidly screening such libraries. Combinatorialpolymer libraries can comprise, for example, reaction product mixturesresulting from reactions that are varied with respect to such factors.General aspects of combinatorial approaches for screening a library arediscussed in more detail in connection with the above-identified patentapplications to which the present invention claims priority. Hence, theinvention can be applied to combinatorial chemistry and materialsscience involving polymers and other materials, as well as to moretraditional HPLC applications. As such, the particular applications andsample materials disclosed herein are to be considered exemplary andnon-limiting.

[0026] Parallel HPLC With Post-Separation Treatment

[0027] With reference to FIG. 1A, characterizing a polymer sampleaccording to the present invention using an HPLC system can include (A)preparing the sample (e.g., dilution), (B) injecting the sample into amobile phase of a flow characterization system (e.g., liquidchromatography system, flow-injection analysis system), (C) separatingthe sample chromatographically, (T) treating the sample (e.g., with aprecipitating agent or a derivatizing agent) to change a property (e.g.,an optical property) of the sample or of a separated component thereof,(D) detecting a property of the polymer sample or of a componentthereof, and/or (E) correlating the detected property to acharacterizing property of interest. As depicted in FIG. 1A, variouscharacterization protocols may be employed involving some or all of theaforementioned steps. The HPLC methods of the present invention involveinjection generally include at least sample injection, chromatographicseparation, treatment and detection (steps B, C, T and D).

[0028] Prior art applications of post-separation treatment steps (e.g.,precipitation) were effected in series with preceding separation stepsand with subsequent detection steps in a single chromatographic channel(I). With reference to FIG. 1B, for example, a plurality of polymersamples can be characterized with a single polymer characterizationsystem (I) in a traditional serial approach in which each of theplurality of polymer samples (s₁, s₂, s₃ . . . s_(n)) are processedserially through the characterization system (I) with each of the steps(A, B, C, T, D, E) effected in series on each of the of samples toproduce a serial stream of corresponding characterizing propertyinformation (p₁, p₂, p₃ . . . p_(n)).

[0029] In contrast, the present invention is directed to parallel HPLCprotocols and systems in which at least the chromatographic separationstep (step C) is effected in a parallel manner (or in a staggeredparallel manner, as disclosed, for example, in copending applicationSer. No. 09/410,546, entitled “Parallel High-Performance LiquidChromatography with Serial Injection”, filed Oct. 1, 1999, by Petro etal under Attorney Docket No. 99-79). With reference to FIG. 1C, forexample, a plurality of polymer samples (s₁, s₂, s₃ . . . s_(n)) can becharacterized with two or more separate (or integrated) polymercharacterization systems (or channels) (I, II, III . . . N) in a pureparallel (or for larger libraries, serial-parallel) approach in whichthe plurality of polymer samples (s₁, s₂, s₃ . . . s_(n)) or a subsetthereof are processed through the two or more polymer characterizationsystems (I, II, III . . . N) in parallel, with each individual systemeffecting each step on one of the samples to produce the characterizingproperty information (p₁, p₂, p₃ . . . p_(n)) in parallel.

[0030] In a hybrid, parallel-series approach, certain of the steps ofthe characterization process can be effected in parallel, while certainother steps can be effected in series. Preferably, for example, it maybe desirable to effect the longer, throughput-limiting steps (e.g.,separation) in parallel for the plurality of samples, while effectingthe faster, less limiting steps in series. Such a parallel-series hybridapproach can be exemplified, with reference to FIG. 1D, by parallelsample preparation (step A) of a plurality of polymer samples (s₁, s₂,s₃ . . . s_(n)), followed by serial injection (step B), and thenparallel chromatographic separation, treatment, detection andcorrelation (steps C, T, D and E) to produce a parallel stream ofcorresponding characterizing property information (p₁, p₂, p₃ . . .p_(n)). In another exemplary parallel-series hybrid approach,represented schematically in FIG. 1E, a plurality of polymer samples(s₁, s₂, s₃ . . . s_(n)) are prepared and injected in series into themobile phase of four or more liquid chromatography characterizingchannels (I, II, III . . . N), and then separated, treated, detected andcorrelated in a slightly offset (staggered) parallel manner to producethe characterizing property information (p₁, p₂, p₃ . . . P_(n)) in thesame staggered-parallel manner. If each of the separation and detectionsystems has the same processing rates, then the extent of the paralleloffset (or staggering) will be primarily determined by the speed of theserial preparation and injection. In a variation of the precedingexample, with reference to FIG. 1F, where the detection and correlationsteps are sufficient fast, a plurality of polymer samples (s₁, s₂, S₃ .. . s_(n)) can be characterized by serial sample preparation andinjection, staggered-parallel chromatographic separation and treatment(steps C and T), and then serial detection and correlation, to producethe characterizing property information (p₁, p₂, p₃, . . . p_(n)) inseries. In this case, the rate of injection into the various separationcolumns is preferably synchronized with the rate of detection. In anadditional variation of the preceding example, with reference to FIG.1G, a plurality of polymer samples (s₁, s₂, s₃ . . . s_(n)) can becharacterized by serial sample preparation and injection,staggered-parallel chromatographic separation (step C), and then serialtreatment, detection and correlation (steps T, D and E), to produce thecharacterizing property information (p₁, p₂, p₃ . . . p_(n)) in series.

[0031] The parallel and parallel-hybrid approaches, with post-separationtreatment, can be used with one or more of the several rapid-serialoptimization approaches—directed toward optimization of one or morecharacterization steps (e.g., steps (A) through (E) of FIG. 1A) withrespect to speed and quality of information—that are disclosed in theabove-identified patent applications to which the instant applicationclaims priority.

[0032] The parallel HPLC system of the present invention comprises adetection system that includes means for treating sample components inparallel chromatographic column eluants. With reference to FIG. 2Athrough FIG. 2C, for example, such detection system can include one ormore treatment systems 30 comprising a treatment agent reservoir 34, atreatment pump 36 and a mixer such as an in-line mixer 32 withappropriate connecting conduits and valving to provide fluidcommunication between the treatment agent reservoir 34 and the mixer 32.As shown in FIG. 2A, the HPLC system can include parallel treatmentsystems for each parallel chromatographic channel. Specifically, two ormore treatment systems 30 a, 30 b, 30 c, 30 d that are each dedicated toa particular chromatographic channel (with each channel including one ormore column(s) 102 a, 102 b, 102 c, 102 d). Parallel treatment forparallel chromatographic channels can also be acheived, with referenceto FIG. 2B, in an alternative embodiment in which the treatment system30 can include one treatment agent reservoir 34 with associatedtreatment pump 36 serving two or more mixers 32 a, 32 b, 32 c, 32 d withappropriate flow-splitters 37 (e.g., “T”-connectors), conduits andvalving to provide fluid communication between the treatment agentreservoir and the two or more mixers 32. According to another approach,with reference to FIG. 2C, the sample components of parallelchromatographic channel eluants can be treated in series, with a singlemixer 32, by routing the two or more channel eluants through a detectionswitching valve 60 for selective, serial delivery to the mixer 32.Briefly, the detection switching valve 60 will have two or moreselectable inlet ports, 62 a through 62 h, and at least one outlet port64. The inlet ports 62 are in fluid communication with two or morechromatography channels (columns), and additionally, are selectable influid communication with the outlet ports 64. Switch 66 can be used toselectively connect one of the inlet ports 62 with the outlet port 64.The outlet port 64 is itself in fluid communication with the mixer 32 ofthe treatment system 30. The switch 66 of the detection valve 60 can bemanually or automatically actuated, and is preferably undermicroprocessor 122 (134) control. The mixer 32 can be supplied with oneor more treatment agents from one or more treatment reservoirs 34 by oneor more treatment pumps 36.

[0033] The post-separation treatment of the present invention can be anytreatment that changes a property of at least one of the separatedsample components of the first and second samples. Preferably, thetreatment is selective to one or more particular sample components ofinterest. Precipitation and derivitization are preferred post-separationtreatment protocols. Preferably, the sample components of the first andsecond (or more) samples are precipitated and/or derivatized—afterseparation but before detection—to make them more susceptible or to makethem selectively detectable to detection, and most preferably, tooptical detection.

[0034] Post-separation precipitation can be effected by any suitablemeans. The treatment can be effected, for example, by combining thechromatographic eluant with a treatment agent—such as a precipitatingagent or derivatizing agent (e.g., in a mixer). Preferably, thecomposition, flow-rate and/or temperature of the post-separation columneluant is controlled to selectively precipitate the one or more samplecomponents of interest. Most preferably, a non-solvent for the componentof interest is combined with the column eluant at various flowrates forselective precipitation. See Examples 1 through 4. See also EuropeanPatent EP 675 356 B 1, which is incorporated by reference in itsentirety for all purposes.

[0035] Derivitization can include any type of derivitizing chemicalreaction known in the art that results in a product that has propertiesdifferent from the reactant sample component. The selective oxidation ofan alcohol to the corresponding ketone is exemplary. Other derivitizingagents are known in the art for incorporating markers, labels (e.g.,fluorescent compounds, radioactive elements or compounds, dyes, etc.)

[0036] Advantageously, such protocols can be cost-effectively applied incombination with-parallel optical-detectors and moreover, suchcombination can be efficiently and suitable applied in mini- andmicro-scaled liquid chromatography systems. As noted below, such mini-and micro-scale liquid chromatography systems can be advantageouslyapplied in connection with combinatorial chemistry and materials scienceresearch.

[0037] The particular configuration for the parallel chromatographicseparation and/or parallel chromatographic channels with which theaforementioned post-separation treatment protocols/treatment systems areemployed is not narrowly critical. In general, the parallelchromatography can include pure parallel configurations as well ashybrid parallel-series configurations, as described above. As usedherein, parallel chromatographic separation/channels means thatchromatographic separation of at least two or more samples is effectedin at least two or more channels, respectively. The separation is atleast partially overlapped (simultaneous separation), althoughinitiation and conclusion times may differ. Additionally, andparticularly as applied to combinatorial chemistry and materials scienceapplications, parallel chromatographic separation of a subset of thetotal number of samples being evaluated can be particularlyadvantageous.

[0038]FIG. 3A shows one embodiment of a parallel chromatography system.The system comprises two or more chromatographic columns 102 a, 102 b,102 c, 102 d into which a mobile phase is supplied in parallel throughan injection valve 100 a, 100 b, 100 c, 100 d, respectively, by HPLCpump 116 from a mobile phase reservoir 114, via appropriateflow-splitters, conduits and valving. A plurality of samples can beloaded into the injection valves 100 through sample loading ports 204 a,204 b, 204 c, 204 d. The samples can be loaded in parallel, oralternatively, in a stepped serial fashion. In operation, a mobile phaseis supplied in parallel from the reservoir 114 to each of the columns102, and samples are injected into the mobile phases through injectionvalves 100. Following injection, the injected samples are optionallyfiltered with in-line filters 104 and one or more sample components arethen chromatographically separated from the sample and/or from othersample components thereof. The separated samples/sample components canthen be delivered to the detection system for treatment and subsequentdetection (e.g., according to one of the embodiments discussed above).

[0039] One embodiment of a preferred hybrid parallel-serialchromatographic separation approach and system (with serial injection)isshown FIG. 3B, and discussed in greater detail in co-pending U.S. patentapplication Ser. No. ______ entitled “Parallel High-Performance LiquidChromatography With Serial Injection” filed by Petro et al. on the dateeven herewith under Attorney Docket No. 99-79. Briefly, the parallelHPLC system with serial injection 10 can comprise a sample injectionsystem 90 for serially and distributively injecting a plurality ofsamples into a liquid mobile phase supplied in parallel to each of twoor more chromatographic columns 102 a, 102 b, 102 c, 102 d. Theinjection system comprises an injector and a multi-port switching valve.The injector, which is preferably an injection valve 100 such as aretypically employed in single-channel HPLC systems, provides a motiveforce for injecting a sample under pressure into the mobile phases beingsupplied to the two or more chromatographic columns. The multi-portswitching valves provides sequential distribution of the samples to themobile phases. At least one sample component of the plurality ofinjected samples are separated from other sample components thereof inthe respective chromatographic columns 102 a, 102 b, 102 c, and 102 d,and a property of at least one of the separated sample components istreated in the mixer 32 a, 32 b, 32 c, 32 d, and then detected in one ormore flow-through detectors 130 a, 130 b, 130 c, 130 d. The liquidmobile phase is supplied in parallel to the chromatographic columns 102a, 102 b, 102 c, 102 d from a mobile-phase source through two or morecolumn supply conduits 80 a, 80 b, 80 c, 80 d, which can also in-linepressure reducers 82 a, 82 b, 82 c, 82 d (e.g., flow restrictors),respectively, and in-line injection connectors 84 a, 84 b, 84 c, 84 d,respectively. The reservoir(s) 114 can be of any suitable design andcapacity, and typically have a volume of about 4 liters. The one or morepumps 116 can be of any type and size suitable to provide a motive forcefor the mobile-phase fluid through the systems 10. After passing throughthe chromatographic columns 102 a, 102 b, 102 c, 102 d and detectors 130a, 130 b, 130 c, 130 d, the mobile phase is discharged from the systemvia a common discharge header and effluent port 141 into a wastecollection container 140. Variations in this embodiment, as well asother embodiments for such parallel-serial approach are disclosed in thereferenced co-pending application.

[0040] Regardless of the particular configuration of the parallelchromatographic system and/or of the treatment and detection systems,the internal system pressures (e.g., mobile phase pressures) deliveredby the HPLC pump are typically at least about 100 psig, at least about200 psig, at least about 500 psig or at least about 1000 psig. Higherpressures, up to several thousand psig, can also be employed in robustsystems. Hence, the pump pressures can range from about 100 psig toabout 6000 psig, from about 200 psig to about 4000 psig, from about 500psig to about 4000 psig, and from about 1000 psig to about 4000 psig.Typical high-pressure liquid chromatography pumps, availablecommercially from various sources, such as Waters Model No. 515(Milford, Mass.) can be employed. The one or more pumps 116 can becontrolled with one or more microprocessors 134. In operation, pumppressures can vary substantially depending on the particularconfiguration of the system 10, including for example the number ofchromatographic columns 102, the separation media employed therein, thedesired flowrates, the desired robustness, etc.

[0041] With reference to FIG. 4, a preferred injection valve 100 for usein connection with the parallel HPLC systems of the invention can be an8-port, two-loop injection valve 210 (100) that operates as follows. Afirst sample is loaded directly into an injection port 108 or indirectlythrough a loading port 204, transfer line 206 and the injection port 108at relatively low pressure compared to the pressure of the mobile phase.The loading port 204 can be adapted in size to accommodate one or moreinjection probes (tips) of a manual or an automated sample delivery unit(e.g., an auto-sampler). When the 8-ported valve is in valve position“A” (with internal flow-paths for the valve indicated by solid lines),the first sample is loaded into a sample loop 205A while the mobilephase flows through the valve via mobile-phase inlet port 101 (theflow-in port), sample loop 205B, and mobile-phase outlet port 103 (theflow-out port). The sample loops 205A and 205B can be of equal volume orof varying volume. A waste port 207 can be employed for receiving anyoverflow sample and/or for flushing the valve after each sample, ifnecessary. When the injection valve 210 is switched to the valve “B”position (with internal flow-paths for the valve now indicated by thedashed lines), the mobile phase then flows through the valve viamobile-phase inlet port 100, sample loop 205A, and mobile-phase outletport 103, and the first sample is thereby injected, via the multi-portswitching valve, into the mobile phase of one of the chromatographiccolumns 202 of the liquid chromatography system 10. The mobile phaseoutlet port 103 is the sample discharge port of the injection valve whena sample is present in the mobile phase. While the first sample is beinginjected from sample loop 205A into the first mobile phase, a secondsample can be loaded into sample loop 205B, ready to be injected oncethe injection valve 100 is switched back to valve position A, and themulti-port switching valve is switched to provide a path of fluidcommunication to the mobile phase of a second chromatographic column.Eight-ported valves, such as represented in FIG. 3, can be purchasedfrom Valveco Instruments Co. Inc. (Houston, Tex.), and the purchasedvalve fittings can be modified as described above for use in connectionwith a flow characterization system. An eight port injection valve 210is a preferred injection valve 100 because the two sample loops 205A,205B allow the flow characterization system to be ready for sampleloading at all times (i.e., has a load/load capability). As such, theeight-port valve is faster than, for example, a six port valve (e.g., avalve having only a single load position and a single inject position),and therefore, the eight-port injection valve provides one aspect forimproving the sample throughput for a liquid chromatography system 10 ora flow-injection analysis system 20. While the eight-port valve 210depicted schematically in FIG. 4 is a preferred configuration, otherhigh-pressure injection valves can also be suitably employed, including,without limitation, valves having a greater or lesser number of ports.Typically, however, a high-pressure injection valve will have from 6 to24 ports.

[0042] While the aforementioned embodiment is preferred, the particulardesign of the injection valve is not critical. The injection valve 100(210) can be configured, for example, to have more than one injectionport 108, a single injection port 108, and in either case, the single ormultiple injection ports 108 be in fluid communication with a number ofloading ports 204 via a number of transfer lines 206 in order to receivesamples independently from a number of different injection probes,including, for example, a manual injection probes, and one or moreprobes associated with automated delivery systems, such as one or morerobotic auto-samplers. The injection valve can also have a larger numberof sample loops with the same or with varying volumes, to accommodatedifferent samples sizes.

[0043] Sample loading into the injection system, also referred to hereinas “sampling”, can be effected in any suitable manner, and theparticular manner employed is not critical to the invention. Sampling ofa sample generally refers to a plurality of steps which includewithdrawing a polymer sample from a sample container and delivering atleast a portion of the withdrawn sample to the injection system of theHPLC system. Sampling may also include additional steps, particularlyand preferably, sample preparation steps. (See FIG. 1A). In oneapproach, only one sample is withdrawn into the auto-sampler probe at atime and only one sample resides in the probe at one time. The onesample is expelled therefrom (for sample preparation and/or into thepolymer characterization system) before drawing the next sample. In analternative approach, however, two or more samples can be withdrawn intothe auto-sampler probe sequentially, spatially separated by a solvent,such that the two or more samples reside in the probe at the same time.Such a “candystriping” approach can provide for very high auto-samplerthroughputs for rapid introduction of the one or more samples into theflow characterization system.

[0044] The sample container from which the polymer sample is withdrawnis not critical. The sample container can be, for example asample-containing well. The sample-containing well can be a sample vial,a plurality of sample vials, or a sample-containing well within an arrayof sample-containing wells (e.g., constituting a polymer samplelibrary). The sample container can alternatively be a sample port from asample line in fluid communication with an industrial process line, suchas a polymerization process line.

[0045] In the general case, sampling can be effected manually, in asemi-automatic manner or in an automatic manner. A polymer sample can bewithdrawn from a sample container manually, for example, with a pipetteor with a syringe-type manual probe, and then manually delivered to aloading port or an injection port of a polymer characterization system.In a semi-automatic protocol, some aspect of the protocol is effectedautomatically (e.g., delivery), but some other aspect requires manualintervention (e.g., withdrawal of polymer samples from a process controlline). Preferably, however, the polymer sample(s) are withdrawn from asample container and delivered to the characterization system in a fullyautomated manner—for example, with an auto-sampler.

[0046] A plurality of samples, such as those included within a libraryof samples, is preferably delivered to the injection system (e.g., toinjection valve 100 in FIG. 3A) loading into the HPLC system, with anautomatic delivery device, such as an auto-sampler. As used herein, theterm “auto-sampler” refers to an apparatus suitable for automatedsampling of polymer samples for characterization, including automatedwithdrawal of a polymer sample from a sample container, and automatedloading of at least a portion of the withdrawn sample into an injectionport or a loading port of a flow characterization system (e.g. a liquidchromatography system).

[0047] Automated sampling equipment is available commercially forintroducing multiple samples into liquid flow systems in a serialmanner. For example, autosamplers that can be suitably adapted for usein connection with the present invention are available from Gilson.While such commercially-available auto-sampling equipment could be usedwith this invention, improved autosamplers as disclosed in copendingU.S. patent application Ser. No. 09/285,393 entitled “Automated SamplingMethods for Rapid Characterization of Polymers”, filed Apr. 2, 1999 byPetro et al. are preferably employed. Such autosamplers providehigh-throughput, with substantial flexibility with respect to samplepreparation, etc., and as such, are well suited to applications of thepresent invention to combinatorial materials science research.

[0048] Briefly, with reference to FIG. 6, in a preferred embodiment anauto-sampler 200 can comprise a movable probe (tip) 201, typicallymounted on a support arm 203, a translation station 221 for providingthree-dimensional motion of the probe, and a microprocessor 222 forcontrolling three-dimensional motion of the probe between variousspatial addresses. The auto-sampler 200 preferably also comprises auser-interface (not shown) to allow for user programming of themicroprocessor 222 with respect to probe motion and manipulations. Theprobe 201 can have an interior surface defining a sample-cavity and aninlet port for fluid communication between the sample cavity and apolymer sample 20. The probe 201 is also adapted for fluid communicationwith an injection port 108 (FIG. 2A, FIG. 2B) or a loading port 204(FIG. 2A, 2B) of the injection system 90. The support arm 203 ispreferably an XYZ robotic arm, such as cane be commercially obtainedfrom Cavro Scientific Instruments, Inc. (Sunnyvale, Calif.) amongothers. To improve smoothness of operation at high speeds, such XYZrobotic arms preferably have motions based on gradient variations ratherthan step-function variations, and preferably are belt driven ratherthan shaft driven. The microprocessor 222 can be a computer and can bethe same or different from the microprocessor 134 (FIG. 2A, FIG. 2B)used to control the detectors 130 (FIG. 2A, FIG. 2B) and dataacquisition therefrom. The auto-sampler can further comprise one or morepumps (not shown), preferably syringe pumps, for drawing and/orexpelling liquids, and related connection lines (not shown) for fluidcommunication between the pumps, the probe 201, and liquid (e.g.solvent) reservoirs. Preferred embodiments include two or more syringepumps—one with a relatively lower flowrate capacity and one with arelatively higher flowrate capacity. Alternative pump configurations,such as peristaltic pumps, vacuum-pumps or other motive-force providingmeans can be used additionally or alternatively. Sampling throughputsmay also be enhanced by using two or more robotic arms together. It islikewise possible to have more two or more sample probes in connectionwith a single robotic arm—for example, such as an array of two or moreprobes each capable of synchronized motion relative to each other.

[0049] In operation, the microprocessor 222 of the auto-sampler 200 canbe programmed to direct the auto-sampler 200 to withdraw a sample 20(e.g., a polymer solution comprising a dissolved polymer) from a samplecontainer (e.g., a sample well) formed in a sample tray 202 into theinjection probe 201, and subsequently to direct the probe 201 to theloading port 204 for loading the sample into the characterization systemthrough transfer line 206. In preferred embodiments, the auto-samplercan be programmed to automatically sample each well of a library ofsamples one after the other whereby a plurality of samples are seriallyloaded into the flow characterization system, and subsequently seriallyinjected into the mobile phase of the characterization system in a plugflow fashion. Preferably, the microprocessor 222 of the auto-samplercomprises a user-interface that can be programmed to allow forvariations from a normal sampling routine (e.g., skipping certainelements at certain spatial addresses of a library). The auto-sampler200 can also be controlled for manual operation on an individual sampleby sample basis.

[0050] The microprocessor 222 is also preferably user-programmable toaccommodate libraries of polymer samples having varying arrangements ofarrays of polymer samples (e.g., square arrays with “n-rows” by“n-columns”, rectangular arrays with “n-rows” by “m columns”, roundarrays, triangular arrays with “r” “r” equilateral sides, triangulararrays with “r-base” by “s-” by “s-” isosceles sides, etc., where n, m,r, and s are integers). More particularly, for example, with respect tosquare or rectangular arrays, a two sets of samples (e.g., libraries)having different spatial configurations can be sampled as follows.First, an auto-sampler is programmed (e.g., via a user interface module)with location information for a first set of samples comprising aplurality of samples in a plurality of sample containers in firstspatial arrangement (e.g., “n-rows” by “m-columns”, where n and m areintegers). The first set of samples are serially withdrawn from theirrespective sample containers, and at least a portion of each of thewithdrawn first set of samples are serially delivered to thesample-loading port of the injection system. The auto-sampler is thenreprogrammed with location information for a second set of liquidsamples that comprise a plurality of samples in a plurality of samplecontainers in second spatial arrangement (e.g., “p-rows” by “q-columns”,where p and q are integers). The second set of samples are seriallywithdrawn from their respective sample containers, and at least aportion of each of the withdrawn second set of samples are seriallydelivered to the sample-loading port of the injection system.

[0051] In a preferred protocol for sampling a plurality of polymersamples, an auto-sampler provides for rapid-serial loading of theplurality of polymer samples into a common injection port of aninjection valve. More specifically, a plurality of polymer samples issampled as follows. At a first withdrawal time, t_(ASW1), a firstpolymer sample is withdrawn from a first sample container at a firstlocation into a probe of an auto-sampler. At least a portion of thewithdrawn first sample is then delivered to an injection port of apolymer characterization system, either directly, or through a loadingport and a transfer line. After delivery of the first polymer sample, asecond polymer sample is, at a second withdrawal time, t_(ASW2),withdrawn from a second sample container at a second location into theauto-sampler probe. At least a portion of the withdrawn second sample isthen delivered (directly or indirectly) to the sample-loading port(e.g., injection port). The cycle can then be repeated, as necessary, inan automated manner, for additional samples included within theplurality of samples.

[0052] The auto-sampler cycle time, T_(AS), delineated by the differencein time, t_(ASW2)−t_(ASW1), is not critical, and can vary widelydepending on the application of the present invention. If the parallelchromatography techniques of the present invention are applied inconnection with standard, conventional HPLC systems and protocols(typically involving from about 30 minutes to about 60 minutes or moreper sample), the sampling cycle time, T_(AS), can range from about tenseconds to about 30 minutes or more. If, however, the parallelchromatography techniques of the invention are applied in connectionwith rapid-serial HPLC systems and protocols as disclosed in theabove-identified co-pending applications from which the presentapplication claims priority, (typically involving from about less than 1minute per sample to about 10 minutes per sample), the sampling cycletime, T_(AS), can range from about ten seconds to about 4 minutes ormore. In general, the sampling time, T_(AS), is preferably not more thanabout 10 seconds, not more than about 15 seconds, not more than about 20seconds, not more than about 30 seconds, not more than about 1 minute,not more than about 2 minutes, not more than about 4 minutes, not morethan about 8 minutes, not more than about 10minutes, not more than about20 minutes, or not more than about 30 minutes.

[0053] The preferred protocol for sampling a plurality of polymersamples can also include additional automated steps, as described in theabove-identified cases from which the present application claimspriority. In particular, sample preparation steps can be incorporatedinto the sampling routine. Such preparation steps can generally beeffected in series with the sample loading/injection steps (See, forexample, FIG. 1B and 1C), or alternatively, can be effected in parallelwith each other (FIG. 1D).

[0054] Various schemes for the timing of sample loading, injecting anddistributing the serially received samples among the two or morechromatographic columns for parallel separation and subsequenttreatement and detection can be employed. In general, the selection of aparticular scheme can depend on factors such as the type of parallelHPLC configuration with which the treatment systems is employed, whetherthe treatement system is set up as a parallel treatment or serialtreatment, the number of samples being characterized, the chemicaldiversity of samples, the number of parallel chromatographic columns inthe HPLC system of the invention, the size of the columns, theseparation media and separation type (e.g., GPC,precipitation-redissolution, adsorption, etc.), the configuration of thedetector(s), and the detection protocols, among others. As such, aperson of skill in the art will have wide latitude to vary the timing,injecting, optionally distributing, separation, treatment and detectionof the plurality of samples.

[0055] The number of parallel chromatographic channels, each comprisinga one or more chromatography columns in series, can generally be two ormore. The number of parallel chromatographic channels (andchromatography columns) is preferably 4 or more, 8 or more, 12 or more,16 or more, 32 or more, 48 or more, 64 or more, or 96 or more. Asdiscussed above in connection with FIG. 4C, nested multi-port switchingvalves can readily accommodate such large numbers of channels.

[0056] With reference to FIGS. 2A and 2B, the chromatographic channelscan also include in-line filters 104 a, 104 b, 104 c, 104 d and/or pulsedampers (not shown) typically incorporated into the sample supplyconduits 80 a, 80 b, 80 c, 80 d. The in-line filters 104 can be of anysuitable dimensions and mesh size. In one embodiment, effective forscreening and evaluation of polymer samples, filters 104 can retainparticles having a diameter of more than about 0.5 μm. In anotherembodiment for polymer samples, filters 104 can retain particles havinga diameter of more than about 0.2 μm. Other sizes may also be employed,as suitable for a particular sample and/or process application.Additional in-line filters can likewise be employed. While shown inFIGS. 2A and 2B inunediately downstream of the connectors 84 a, 84 b, 84c, 84 d to the injection system 90, the particular location of thefilters is not critical. Moreover, the sample could be filtered as apreparation step, prior to loading of the sample into the HPLC system.Other in-line systems, such as pulse-dampers can also be employed.

[0057] After injection of a sample into a stream of liquid serving as amobile phase of a liquid chromatography channel, the sample isintroduced into a chromatographic column containing a separation mediumhaving a stationary phase for separation of one or more components ofthe sample from other components thereof. Separation is effected byselectively eluting one or more of the components from thestationary-phase with the mobile-phase acting also as an eluant. Thedegree of separation, also referred to as the resolution of the polymersample components, can vary depending on the particular chemical natureof the polymer sample components, and the quality of informationrequired in the particular characterization application. In general, theseparation performance in a given case can be controlled as a functionof the column design /geometry, the stationary phase media, and theelution conditions with the mobile phase.

[0058] The particular design of a chromatographic column for liquidchromatography is, in the general case, not narrowly critical. A numberof columns known in the art can be employed in connection with thepresent invention—as purchased or with minor variations disclosedherein. In general, with reference to FIG. 2A, the chromatographiccolumn 102 of a liquid chromatography system 10 comprises an interiorsurface defining a pressurizable separation cavity having a definedvolume, an inlet port for receiving a mobile phase and for supplying apolymer sample to the separation cavity, and an effluent port fordischarging the mobile phase and the polymer sample or separatedcomponents thereof from the separation cavity. The separation cavity ispreferably pressurizable to pressures typically involved withhigh-pressure liquid chromatography—such pressures generally rangingfrom about atmospheric pressure to about 6000 psig (about 40 MPa). Insome preferred liquid-chromatography characterization methods, discussedin greater detail below, the chromatographic column can be relativelyshorter, and relatively wider, compared to traditional chromatographicseparation columns. Such preferred high-aspect ratio columns aredisclosed in greater detail in co-pending U.S. patent application Ser.No. 09/285,393 entitled “Rapic Characterization of Polymers”, filed Apr.2, 1999 by Petro et al.

[0059] The chromatographic column 102 further comprises a separationmedium having a stationary-phase within the separation cavity. Theseparation medium can consist essentially of a stationary-phase or canalso include, in addition thereto, an inert support for the stationaryphase. The column 102 can also comprise one or more fillers, frits (forseparation medium retention and/or for filtering), and various fittingsand features appropriate for preparing and/or maintaining the column forits intended application. The particular separation medium to beemployed as the stationary-phase is not critical, and will typicallydepend on the separation strategy for the particular chemistry of thepolymer samples of interest, as well as on the desired detection,sample-throughput and/or information quality. Typical stationary-phasemedia can be a bed of packed beads, rods or other shaped-particles, or amonolithic medium (typically greater than about 5 mm in thickness), eachof which can be characterized and optimized for a particular separationstrategy With respect to the material, size, shape, pore size, pore sizedistribution, surface area, solvent regain, bed homogeneity (for packedshaped particles) inertness, polarity, hydrophobicity, chemicalstability, mechanical stability and solvent permeability, among otherfactors. Generally preferred stationary-phase include porous media(e.g., porous beads, porous monoliths), such as are suitable for gelpermeation chromatography (GPC), and media suitable forprecipitation-redissolution chromatography, adsorption chromatography,and/or reverse-phase chromatography. Non-porous particles or emptycolumns and/or capillaries with adsorptive walls can be used as well. Ifbeads are employed, spherical beads are preferred over other shapes.Particularly preferred stationary-phase media for polymercharacterization applications are disclosed in greater detail below, butcan generally include silica, cross-linked resins, hydroxylatedpolyglycidyl methacrylates,(e.g.,poly(2-3-dihydroxypropylmethacrylate)), poly(hydroxyethyl methacrylate),and polystyrenic polymers such as poly(styrene-divinylbenzene).

[0060] The mobile-phase fluid(s) employed to elute one or more polymercomponents from a chromatographic stationary-phase are not generallycritical, and can vary depending on the chemistry of the separationbeing effected. The mobile phase can be varied with respect tocomposition, temperature, gradient rates, flow-rates, and other factorsaffecting selectivity, speed of separation, peak capacity (e.g., maximumnumber of components that can be separated with a single run) and/orresolution of a polymer component. Exemplary mobile-phase fluids for GPCinclude tetrahydrofuran (THF), toluene, dimethylformamide, water,aqueous buffers, trichlorobenzene and dichlorobenzene. Exemplarymobile-phase fluids for precipitation-redissolution chromatographyinclude THF, methanol, hexane, acetone, acetonitrile and water. Foradsorption chromatography, the mobile phase can include, for example,hexane, isooctane, decane, THF, dichloromethane, chloroform,diethylether and acetone. For reverse-phase chromatography, the mobilephase can include water, acetonitrile, methanol and THF, among others.

[0061] Significantly, preferred mobile phase flow are typically fasterthan flowrates employed conventionally for high-pressure liquidchromatography. The flowrates can vary, depending on the separationbeing effected, but can, in many instances, range from about 0.1 ml/minabout 25 ml/min, and preferably range from about 1 ml/min to about 25ml/min. It may be desirable, for some detector configurations, to splitoff a part of the sample-containing mobile phase such that the flow rateto a particular detector is reduced to an acceptable level. For liquidchromatography systems, such a split would typically occur after thecolumn and before the detector.

[0062] A treated sample such as a treated polymer sample (or one or morecomponents thereof) is characterized by detecting a property of thesample, or by detecting a property of a component (e.g., a polymercomponent, a monomer component) of the sample. In many cases, theproperty is detected over a period of time, such that a variation in theproperty can be observed or detected or the rate of change of variationof a property can be observed or detected. In the general case, thedetected property call be any property which can provide ascientifically meaningful basis of comparison between two differentpolymer samples or between two different polymer components—eitherdirectly, or after being correlated to a specific characterizingproperty of interest. The detected property can be a chemical propertyor a physical property of the sample or component thereof. In preferredapplications, an optical property of the polymer sample or a componentthereof can be detected. For example, an amount, frequency, intensity ordirection of an incident light that is refracted, scattered, and/orabsorbed by the polymer sample or a component thereof may be detected.Other properties, such as pressure or other factors affecting aparticular characterizing property of interest (e.g., viscosity) canlikewise be detected.

[0063] The detection step can be performed in parallel, inserial-parallel, or in series. With reference to FIGS. 2B and 2C, aproperty of a sample or of a component thereof, such as achromatographically separated component thereof, can be detected withone or more detectors 130.

[0064] Parallel detection can be effected with two or more detectors(e.g., detectors 130 a, 130 b, 130 c, 130 d as shown in FIGS. 2A, 2B),and with each of such detectors being dedicated to one or morechromatographic channels (i.e., the flow cells of each of such detectorsbeing in fluid communication with one or more chromatography columns).Parallel detection is particularly preferred in combination withrapid-serial techniques (e.g., overlaid injection/separation techniques)applied to any particular chromatographic column. In one preferredparticular approach, parallel flow cells—each being dedicated to onchromatographic channel—are employed but the detection electronicsassociated therewith is electronically and serially switched between twoor more of the flow cells, thereby reducing the amount of analysiscircuitry required.

[0065] Serial detection can also be effected, particularly wheredetection is faster than the separation, and within the timing intervalsfor sampling, injection and switching. In one serial embodiment, shownin FIG. 2C, the parallel chromatography column effluents (e.g.,mobilephase w/separated samples) can be serially directed through a detectionswitching valve 60 to the flow-cell 131of a detector 130.

[0066] In preferred embodiments, a property of a polymer sample or of acomponent thereof is detected with an optical detector such as arefractive-index detector, an ultraviolet-visual detector, a photodiodearray detector, a static-light-scattering detector, adynamic-light-scattering detector, and/or anevaporative-light-scattering detector—also known as an evaporative massdetector (EMD). Other detectors (e.g., a capillary viscometer detector,photodiode array detector (PDAD), infra-red detector, fluorescencedetector, electrochemical detector, conductivity detector, etc.) canlikewise be employed in connection with the present invention. Theparticular nature of the detector (e.g., shape and/or configuration of adetection cavity 131 within the detector) is not generally critical.

[0067] The protocols for characterizing one or more samples preferablyfurther comprise determining a property of interest from the detectedproperty. The physically-detected properties, such as the capability ofthe-sample or component thereof to refract, scatter, emit or absorblight can be correlated to properties of interest. For polymer samples,for example, such properties of interest include, without limitation,weight-average molecular weight, number-average molecular weight,viscosity-average molecular weight, peak molecular weight, approximatemolecular weight, polydispersity index, molecular-weight-distributionshape, relative or absolute component concentration, chemicalcomposition, conversion, concentration, mass, hydrodynamic radius (Rh),radius of gyration (R_(g)), chemical composition, amounts of residualmonomer, presence and amounts of other low-molecular weight impuritiesin polymer samples, particle or molecular size, intrinsic viscosity,molecular shape, molecular conformation, and/or agglomeration orassemblage of molecules. The correlation between a detected property anda determined property of interest can be based on mathematical modelsand/or empirical calibrations. Such correlation methods are generallyknown in the art, and are typically incorporated intocommercially-available chromatographic detectors and/or detector ordata-acquisition software.

[0068] For combinatorial polymer science research applications, as wellas other applications, the characterization protocols can be effected todetermine at least a weight-average molecular weight as acharacterization property of primary importance. Other characterizationproperties of interest of substantial importance, include number-averagemolecular weight, polydispersity index, andmolecular-weight-distribution shape. For polymer samples that arepolymerization product mixtures, another characterization property ofsubstantial importance is conversion data for the polymerizationreaction, typically expressed as % monomer converted into polymer. Thecomposition of the polymer sample or of particular components thereof(e.g., polymer components) can also be of substantial importance.

[0069] For determining weight-average molecular weight from detectedproperties, a liquid chromatography system or a flow-injection analysissystem can advantageously employ a single detector or a combination oftwo or more detectors. In a single-detector embodiment, for example, adynamic light-scattering (DLS) detector can be used by itself todetermine an average hydrodynamic radius or a distribution ofhydrodynamic radii from the detected scattered light. The hydrodynamicradii can, in turn, be correlated to an average molecular weight or amolecular weight distribution. In a two-detector embodiment, forexample, a static-light scattering (SLS) detector (where the detectedscattered light is a function of weight-average molecular weight(M_(W)), concentration (C) and the square of the refractive indexincrement, (dn/dC)²) can be combined with a refractive index (RI)detector (where the detected refracted light is a function of (C) and(dn/dC)), with an ultraviolet/visible light absorbance (UV/VIS) detector(where the detected absorbed light is a function of (C)), or with anevaporative light scattering detector (ELSD) (where the detectedscattered light is a function of (C)). In another embodiment, asingle-detector or multiple detectors (e.g., SLS) can detect theintensity of light scattered by the sample or sample component at two ormore different angles, which can be correlated to molecular weight.

[0070] For polymer samples that are polymerization product mixtures,conversion data or the polymerization reaction of which the sample isrepresentative can be determined by chromatographically resolving thepolymer component(s) and monomer component(s), determining amolecular-weight distribution for such components, integrating areasunder the respective peaks, and then comparing the integrated peak areas(e.g., using response factors for particular components and detectoremployed). Another approach for calculating conversion involvesconverting the polymer-peak area into polymer concentration or massusing a concentration-detector response calibration plot, and thencomparing the portion of the polymer mass or concentration found in thesample to the expected mass or concentration assuming 100%stoichiometric conversion. Composition data for a polymer sample can bedetermined from the consumption of monomer or comonomers or,alternatively, from a retention time per volume of the polymer peak or afraction thereof.

[0071] Advantageously, an ELSD detector, or other detectors that are notparticularly sensitive to low-molecular weight components of a polymersample, can be advantageously employed in connection with the flowcharacterization protocols of the invention to achieve a highsample-throughput. As discussed in greater detail below, detectors thatare insensitive to low-molecular weight components can be advantageouslyemployed in connection with rapid-serial overlapping techniques.Moreover, because the ELSD is also less sensitive to temperaturevariations than other types of mass detectors (e.g., RI detector) and isnot required to be in thermal equilibrium with the sample beingdetected, an ELSD detector can be employed advantageously in connectionwith high-temperature polymer characterization systems. Hence, detectinga property of a polymer sample or a component there of with an ELSD orwith other low-MW insensitive or less temperature sensitive massdetectors provides a further aspect for improving the samplethroughput—particularly for a liquid chromatography system 10 or aflow-injection analysis system 20.

[0072] The aforementioned characterizing properties of interest can,once determined, be mathematically combined in various combinations toprovide figures of merit for various properties or attributes ofinterest. In particular, for example, molecular weight, conversion andpolydispersity index can be evaluated versus polymerization process timeto provide mechanistic insights as to how polymers are formed. Othercombinations of the fundamental characterization properties of interestwill be apparent to those of skill in the art.

[0073] Specific applications and/or combinations of detectors, as wellas correlation protocols, are discussed in greater detail in theabove-identified co-pending U.S. applications to which the presentapplication claims priority.

[0074] Referring to the various figures, one or more microprocessorscan, as noted above, be employed for controlling every aspect of theflow characterization systems, including: the pump 116 (e.g.,mobile-phase flow-rate, flow-rate gradients, compositional gradients,temperature gradients, acceleration rates for such gradients); thereservoir 114 (e.g., temperature, level); the auto-sampler 200 (e.g.,movements between spatial position, timing thereof, sample selection,sample preparation, sampling pump flow-rates, and other operations), theinjection valve 100 (e.g., timing, selection of sample loops, etc.); themulti-port switch 70, the column 102 (e.g., column selection (ifmultiple columns and automated column-switching valves are present),column temperature); the detection switch 60 (as applicable), thedetector 130 (e.g., data acquisition (e.g., sampling rate), dataprocessing (e.g., correlation); the detector parameters (e.g.,wavelength); and/or overall system conditions (e.g., system pressure,temperature). Software is typically available from detector and/orliquid chromatography system manufacturers (e.g., MILLENIUM™ 2000software available from Waters (Milford, Mass.).

[0075] Inverse Chromatography and Other Solid-Phase InteractionEvaluations

[0076] In one application, the present invention can be employed,substantially as described above, for “inverse chromatography” studies,in which the object and subject of the study are reversed as compared to“regular” chromatography. In addition, this concept can beadvantageously extended to the study of other solid phase—liquid phaseinteractions (that may not necessarily involve separation of samplecomponents and, as such, may not be considered to be “chromatography”).

[0077] In general, a plurality of samples are serially injected into amobile phase supplied in parallel to two or more columns, where thecolumns comprise solid or supported materials. The solid or supportedmaterials can be separation media, or can be other types of solids forwhich there is an interest to study interactions with a mobile phaseand/or vice versa. The interaction between the injected samples, or oneor more components of the injected samples, and the solid or supportedmaterials in the columns is then evaluated.

[0078] Samples

[0079] In general, the sample materials can generally comprise elementsor compounds selected from the group consisting of organic materials,inorganic materials and metal-ligands. In some applications, thecandidate materials will consist essentially of organic materials,consist essentially of inorganic materials, or consist essentially ofmetal-ligand materials. Moreover, in some applications, the samplematerials will be compositions comprising mixtures of organic materials,inorganic materials and/or metal-ligand materials in the variouspossible combinations.

[0080] Organic materials are considered to include compounds havingcovalent carbon-carbon bonds. In some embodiments, the organic materialsare preferably organic polymers, small-organic molecules having amolecular weight of less than about 1000, or non-biological molecules.Non-biological organic materials include organic materials other thanbiological materials. Biological materials are considered to includenucleic acid polymers (e.g., DNA, RNA) amino acid. polymers (e.g.,enzymes) and small organic compounds (e.g., steroids, hormones) wherethe small organic compounds have biological activity, especiallybiological activity for humans or commercially significant animals suchas pets and livestock, and where the small organic compounds are usedprimarily for therapeutic or diagnostic purposes. Although in someapplications the sample materials being characterized by the HPLC systemare preferably not, themselves, biological organic materials, the samplematerials of the invention (e.g., polymers) can be employed to prepareor separate biological organic materials. Polymeric sample materials arediscussed in greater detail below.

[0081] Inorganic materials include elements (including carbon in itsatomic or molecular forms), compounds that do not include covalentcarbon-carbon bonds (but which could include carbon covalently bonded toother elements, e.g., CO₂), and compositions including elements and/orsuch compounds.

[0082] The samples can comprise materials that are an element, acompound or a composition comprising a plurality of elements and/orcompounds. The sample materials are generally in a liquid state or arecapable of being dissolved, dispersed or emulsified in a liquid phase,as appropriate for chromatographic separation (or, with respect toinverse chromatagraphy, as appropriate for the interaction between thesamples and the solid or supported material.

[0083] The samples can be reaction products from a chemical reaction,which for purposes hereof, means a process in which at least onecovalent bond of a molecule or compound is formed or broken. As such,immunoreactions in which immunoaffinity is based solely on hydrogenbonding or other forces—while chemical processes—are not considered tobe chemical reactions. Reactions that include activation of, breakingand/or formation of H—Si, H—H, H—N, H—O, H—P, H—S, C—H, C—C, C═C, C≡C,C—halogen, C—N, C—O, C—S, C—P, C—B and C—Si bonds are exemplary. Morespecific exemplary chemical reactions from which reaction-productsamples may derive, include, without limitation, oxidation, reduction,hydrogenation, dehydrogenation (including transfer hydrogenation),hydration, dehydration, hydrosilylation, hydrocyanation,hydroformylation (including reductive hydroformylation), carbonylation,hydrocarbonylation, amidocarbonylation, hydrocarboxylation,hydroesterification, hydroamination, hetero-cross-coupling reaction,isomerization (including carbon-carbon double bond isomerization),dimerization, trimerization, polymerization, co-oligomerization (e.g.CO/alkene, CO/alkyne), co-polymerization (e.g. CO/alkene, CO/alkyne),insertion reaction, aziridation, metathesis (including olefinmetathesis), carbon-hydrogen activation, cross coupling, Friedel-Craftsacylation and alkylation, Diels-Alder reactions, C-C coupling, Heckreactions, arylations, Fries rearrangement, vinylation, acetoxylation,aldol-type condensations, aninations, reductive aninations,epoxidations, hydrodechlorinations, hydrodesulfurations andFischer-Tropsch reactions, asymmetric versions of any of theaforementioned reactions, and combinations of any of the aforementionedreactions in a complex reaction sequence of consecutive reactions. Acombinatorial library or array comprising different reaction-productsamples can be formed, for example, as the reaction product from achemical reactions involving a library of diverse catalysts, and/orvariations in reactants, co-reactants, cataloreactants, selectiveblocking moieties, etc. As used herein, the term catalyst is intended toinclude a material that enhances the reaction rate of a chemicalreaction would not substantially proceed in the absence of the catalyst.

[0084] Polymer Samples

[0085] The present invention is particularly preferred in connectionwith the characterization of polymer samples, and especially,combinatorial libraries comprising different polymer samples. Thepolymer sample can be a homogeneous polymer sample or a heterogeneouspolymer sample, and in either case, comprises one or more polymercomponents. As used herein, the term “polymer component” refers to asample component that includes one or more polymer molecules. Thepolymer molecules in a particular polymer component have the same repeatunit, and can be structurally identical to each other or structurallydifferent from each other. For example, a polymer component may comprisea number of different molecules, with each molecule having the samerepeat unit, but with a number of molecules having different molecularweights from each other (e.g., due to a different degree ofpolymerization). As another example, a heterogeneous mixture ofcopolymer molecules may, in some cases, be included within a singlepolymer component (e.g., a copolymer with a regularly-occurring repeatunit), or may, in other cases, define two or more different polymercomponents (e.g., a copolymer with irregularly-occurring orrandomly-occurring repeat units). Hence, different polymer componentsinclude polymer molecules having different repeat units. It is possiblethat a particular polymer sample (e.g., a member of a library) will notcontain a particular polymer molecule or polymer component of interest.

[0086] The polymer molecule of the polymer component is preferably anon-biological polymer. A non-biological polymer is, for purposesherein, a polymer other than an amino-acid polymer (e.g., protein) or anucleic acid polymer (e.g., deoxyribonucleic acid (DNA)): Thenon-biological polymer molecule of the polymer component is, however,not generally critical; that is, the systems and methods disclosedherein will have broad application with respect to the type (e.g.,architecture, composition, synthesis method or mechanism) and/or nature(e.g., physical state, form, attributes) of the non-biological polymer.Hence, the polymer molecule can be, with respect to homopolymer orcopolymer architecture, a linear polymer, a branched polymer (e.g.,short-chain branched, long-chained branched, hyper-branched), across-linked polymer, a cycle polymer or a dendritic polymer. Acopolymer molecule can be a random copolymer molecule, a block copolymermolecule (e.g., di-block, tri-block, multi-block, taper-block), a graftcopolymer molecule or a comb copolymer molecule. The particularcomposition of the non-biological polymer molecule is not critical, andcan include repeat units or random occurrences of one or more of thefollowing, without limitation: polyethylene, polypropylene, polystyrene,polyolefin, polyimide, polyisobutylene, polyacrylonitrile, poly(vinylchloride), poly(methyl methacrylate), poly(vinyl acetate),poly(vinylidene chloride), polytetrafluoroethylene, polyisoprene,polyacrylamide, polyacrylic acid, polyacrylate, poly(ethylene oxide),poly(ethyleneimine), polyamide, polyester, polyurethane, polysiloxane,polyether, polyphosphazine, polymethacrylate, and polyacetals.Polysaccharides are also preferably included within the scope ofnon-biological polymers. While some polysaccharides are of biologicalsignificance, many polysaccharides, and particularly semi-syntheticpolysaccharides have substantial industrial utility with little, if anybiological significance. Exemplary naturally-occurring polysaccharidesinclude cellulose, dextran, gums (e.g., guar gum, locust bean gum,tamarind xyloglucan, pullulan), and other naturally-occurring biomass.Exemplary semisynthetic polysaccharides having industrial applicationsinclude cellulose diacetate, cellulose triacetate, acylated cellulose,carboxymethyl cellulose and hydroxypropyl cellulose. In any case, suchnaturally-occurring and semi-synthetic polysaccharides can be modifiedby reactions such as hydrolysis, esterification, alkylation, or by otherreactions.

[0087] In typical applications, a polymer sample is a heterogeneoussample comprising one or more polymer components, one or more monomercomponents and/or a continuous fluid phase. In copolymer applications,the polymer sample can comprise one or more copolymers, a firstcomonomer, a second comonomer, additional comonomers, and/or acontinuous fluid phase. The polymer samples can, in any case, alsoinclude other components, such as catalysts, catalyst precursors (e.g.,ligands, metal-precursors), solvents, initiators, additives, products ofundesired side-reactions (e.g., polymer gel, or undesired homopolymer orcopolymers) and/or impurities. Typical additives include, for example,surfactants, control agents, plasticizers, cosolvents and/oraccelerators, among others. The various components of the heterogenouspolymer sample can be uniformly or non-uniformly dispersed in thecontinuous fluid phase.

[0088] The polymer sample is preferably a liquid polymer sample, such asa polymer solution, a polymer emulsion, a polymer dispersion or apolymer that is liquid in the pure state (i.e., a neat polymer). Apolymer solution comprises one or more polymer components dissolved in asolvent. The polymer solution can be of a form that includeswell-dissolved chains and/or dissolved aggregated micelles. The solventcan vary, depending on the application, for example with respect topolarity, volatility, stability, and/or inertness or reactivity. Typicalsolvents include, for example, tetrahydrofuran (THF), toluene, hexane,ethers, trichlorobenzene, dichlorobenzene, dimethylformamide, water,aqueous buffers, alcohols, etc. According to traditional chemistryconventions, a polymer emulsion can be considered to comprise one ormore liquid-phase polymer components emulsified (uniformly ornon-uniformly) in a liquid continuous phase, and a polymer dispersioncan be considered to comprise solid particles of one or more polymercomponents dispersed (uniformly or non-uniformly) in a liquid continuousphase. The polymer emulsion and the polymer dispersion can also beconsidered, however, to have the more typically employed meaningsspecific to the art of polymer science—of being aemulsion-polymerization product and dispersion-polymerization product,respectively. In such cases, for example, the emulsion polymer samplecan more generally include one or more polymer components that areinsoluble, but uniformly dispersed, in a continuous phase, with typicalemulsions including polymer component particles ranging in diameter fromabout 2 nm to about 500 nm, more typically from about 20 nm to about 400nm, and even more typically from about 40 nm to about 200 nm. Thedispersion polymer sample can, in such cases, generally include polymercomponent particles that are dispersed (uniformly or nonuniformly) in acontinuous phase, with typical particles having a diameter ranging fromabout 0.2 μm to about 1000 μm, more typically from about 0.4 μm to about500 μm, and even more typically from about 0.5 μm to about 200 μm.Exemplary polymers that can be in the form of neat polymer samplesinclude dendrimers, and siloxane, among others. The liquid polymersample can also be employed in the form of a slurry, a latex, a microgela physical gel, or in any other form sufficiently tractable for analysisas described and claimed herein. Liquid samples are useful in theautomated sample-handling tools that prepare and automatically sampleeach member of a polymer library. Liquid samples also allow the sampleto flow in the chromatographic system or characterization system. Insome cases, polymer synthesis reactions (i.e., polymerizations) directlyproduce liquid samples. These may be bulk liquid polymers, polymersolutions, or heterogeneous liquid samples such as polymer emulsions,latices, or dispersions. In other cases, the polymer may be synthesized,stored or otherwise available for characterization in a non-liquidphysical state, such as a solid state (e.g., crystalline,semicrystalline or amorphous), a glassy state or rubbery state. Hence,the polymer sample may need to be dissolved, dispersed or emulsified toform a liquid sample by addition of a continuous liquid-phase such as asolvent. The polymer sample can, regardless of its particular form, havevarious attributes, including variations with respect to polarity,solubility and/or miscibility.

[0089] In preferred applications, the polymer sample is a polymerizationproduct mixture. As used herein, the term “polymerization productmixture” refers to a mixture of sample components obtained as a productfrom a polymerization reaction. An exemplary polymerization productmixture can be a sample from a combinatorial library prepared bypolymerization reactions, or can be a polymer sample drawn off of anindustrial process line. In general, the polymer sample may be obtainedafter the synthesis reaction is stopped or completed or during thecourse of the polymerization reaction. Alternatively, samples of eachpolymerization reaction can be taken and placed into an intermediatearray of vessels at various times during the course of the synthesis,optionally with addition of more solvent or other reagents to arrest thesynthesis reaction or prepare the samples for analysis. Theseintermediate arrays can then be characterized at any time withoutinterrupting the synthesis reaction. It is also possible to use polymersamples or libraries of polymer samples that were prepared previouslyand stored. Typically, polymer libraries can be stored with agents toensure polymer integrity. Such storage agents include, for example,antioxidants or other agents effective for preventing cross-linking ofpolymer molecules during storage. Depending upon the polymerizationreaction, other processing steps may also be desired, all of which arepreferably automated. The polymerization scheme and/or mechanism bywhich the polymer molecules of the polymer component of the sample areprepared is not critical, and can include, for example, reactionconsidered to be additional polymerization, condensation polymerization,step-growth polymerization, and/or chain-growth polymerizationreactions. Viewed from another aspect, the polymerization reaction canbe an emulsion polymerization or a dispersion polymerization reaction.Viewed more specifically with respect to the mechanism, thepolymerization reaction can be radical polymerization, ionicpolymerization (e.g., cationic polymerization, anionic polymerization),and/or ring-opening polymerization reactions, among others. Non-limitingexamples of the foregoing include, Ziegler-Natta or Kaminsky-Sinnreactions and various copolymerization reactions. Polymerization productmixtures can also be prepared by modification of a polymeric startingmaterials, by grafting reactions, chain extension, chain scission,functional group interconversion, or other reactions.

[0090] The sample size is not narrowly critical, and can generally vary,depending on the particular characterization protocols and systems usedto characterize the sample or components thereof. Typical sample sizescan range from about 0.1 μl to about 1 ml, more typically from about 1μl to about 1000 μl, even more typically from about 5 μl to about 100μl, and still more typically from about 10 μl to about 50 μl. Agenerally preferred sample size for flow characterization systems and,particularly for liquid chromatography, is a sample size of about 20 μl.

[0091] The polymer sample, such as a polymerization product mixture, canbe a raw, untreated polymer sample or can be pretreated in preparationfor characterization. Typical sample preparation steps includepreliminary, non-chromatographic separation of one or more components ofa polymer sample from other components, dilution, mixing and/orredissolution (e.g., from a solid state), among other operations.Preliminary separation methods can help remove large-scale impuritiessuch as dust, coagulum or other impurities. Such separation methods caninclude, for example: filtering (e.g., with a microfilter having poresizes that allow the passage of particles less than about 0.5 μm or 0.2μm); precipitation of polymer components, monomer components and/orother small-molecule components, decanting, washing, scavenging (e.g.,with drying agents), membrane separation (e.g., diafiltration,dialysis), evaporation of volatile components and/or ion-exchange. Thesample is preferably diluted, if necessary, to a concentration rangesuitable for detection. For typical liquid chromatography applications,for example, the sample concentration prior to loading into the liquidchromatography system can range from about 0.01 mg/ml to a neat sample,more typically from about 0.01 mg/ml to about 100 mg/ml, and even moretypically from about 0.1 mg/ml to about 50 mg/ml. More specificconcentration ranges typical for liquid chromatography samples includefrom about 0.1 mg/ml to about 20 mg/ml, and from about 0.5 mg/ml toabout 5 mg/ml. For flow-injection analysis systems, in which the sampleis detected without substantial chromatographic separation thereof, muchmore dilute solutions can be employed. Hence, the concentration canrange from a detectable concentration level (for the particular detectoremployed) up to about 1 mg/ml, or more in some applications. Typicalconcentrations can be about 1×10⁻² wt %, about 1×10⁻³ wt % or about 1×10⁻⁴ wt %. Mixing can be required to increase the uniformity of a polymersample emulsion or dispersion, and/or to integrate one or moreadditional components into the polymer sample. Preparation steps, andparticularly rapid preparation techniques, can be an important aspectfor combinatorial polymer investigations—since polymer samples may besynthesized in a form not ideally suited for immediate characterization.

[0092] Non-Polymer Samples

[0093] Although the primary applications of the present invention aredirected to combinatorial polymer science research and/or, qualitycontrol for industrial polymer synthesis or processing protocols,aspects of the invention can have applications involving non-polymersamples. A non-polymer sample can be a material that comprises anorganic or an inorganic non-polymer element or compound. Oligomers areconsidered to be polymers rather than non-polymers. The non-polymermolecule is, in some cases, preferably a non-biological non-polymerelement or compound. Such non-biological non-polymer elements orcompounds include non-polymer elements or compounds other than thosehaving a well-characterized biological activity and/or a primarycommercial application for a biological field (e.g., steroids, hormones,etc.). More particularly, such non-biological, non-polymer elements orcompounds can include organic or inorganic materials such as pigments,carbon powders (e.g., carbon black), metals, metal oxides, metal salts,metal colloids, metal ligands, etc, without particular limitation.

[0094] Pluralities of Samples/Libraries of Samples

[0095] A plurality of samples such as polymer samples comprises 2 ormore samples that are physically or temporally separated from eachother—for example, by residing in different sample containers, by havinga membrane or other partitioning material positioned between samples, bybeing partitioned (e.g., in-line) with an intervening fluid, by beingtemporally separated in a flow process line (e.g., as sampled forprocess control purposes), or otherwise. The plurality of samplespreferably comprises 4 or more samples, more preferably 8 or moresamples, and even more preferably 10 or more samples. Four samples canbe employed, for example, in connection with experiments having onecontrol sample and three polymer samples varying (e.g., with respect tocomposition or process conditions as discussed above) to berepresentative of a high, a medium and a low-value of the variedfactor—and thereby, to provide some indication as to trends. Eightsamples can provide for additional variations in the explored factorspace. Moreover, eight samples corresponds to the number of parallelpolymerization reactors in the PPR-8™, being selectively offered as oneof the Discovery Tools™ of Symyx Technologies, Inc. (Santa Clara,Calif.). Higher numbers of samples can be investigated, according to themethods of the invention, to provide additional insights into largercompositional and/or process space. In some cases, for example, theplurality of samples can be 15 or more samples, preferably 20 or moresamples, more preferably 40 or more samples and even more preferably 80or more samples. Such numbers can be loosely associated with standardconfigurations of parallel reactor configurations (e.g., the PPR-48™,Symyx Technologies, Inc.) and/or of standard sample containers (e.g.,96-well microtiter plate-type formats). Moreover, even larger numbers ofsamples such as polymer samples can be characterized according to themethods of the present invention for larger scale research endeavors.Hence, the number of samples can be 150 or more, 400 or more, 500 ormore, 750 or more, 1,000 or more, 1,500 or more, 2,000 or more, 5,000 ormore and 10,000 or more polymer samples. As such, the number of samplescan range from about 2 samples to about 10,000 samples, and preferablyfrom about 8 samples to about 10,000 samples. In many applications,however, the number of samples can range from about 80 samples to about1500 samples. In some cases, in which processing of samples usingtypical 96-well microtiter-plate formatting is convenient or otherwisedesirable, the number of samples can be 96*N,where N is an integerranging from about 1 to about 100. For many applications, N can suitablyrange from 1 to about 20, and in some cases, from 1 to about 5.

[0096] The plurality of samples can be a combinatorial library ofsamples. A library of samples comprises of two or more differentsamples, and can be in an array format as spatially separatedsamples—preferably on a common substrate, or-temporally separated —forexample, in a flow system. Candidate samples (i.e., members) within alibrary may differ in a definable and typically predefined way,including with regard to chemical structure, processing (e.g.,synthesis) history, mixtures of interacting components, purity, etc. Thesamples can be spatially separated, preferably at an exposed surface ofthe substrate, such that the array of samples are separately addressablefor sampling into the characterization system and subsequentcharacterization thereof. The two or more different samples can residein sample containers formed as wells in a surface of the substrate. Thenumber of samples included within the library can generally be the sameas the number of samples included within the plurality of samples, asdiscussed above. In general, however, not all of the samples within alibrary of samples need to be different samples. When process conditionsare to be evaluated, the libraries may contain only one type of sample.Typically, however, for combinatorial polymer science researchapplications, at least two or more, preferably at least four or more,even more preferably eight or more and, in many cases most, andallowably each of the plurality of polymer samples in a given library ofpolymer samples will be different from each other. Specifically, adifferent polymer sample can be included within at least about 50%,preferably at least 75%, preferably at least 80%, even more preferablyat least 90%, still more preferably at least 95%, yet more preferablyat-least 98% and most preferably at least 99% of the polymer samplesincluded in the sample library. In some cases, all of the polymersamples in a library of polymer samples will be different from eachother.

[0097] The substrate can be a structure having a rigid or semi-rigidsurface on which or into which the array of polymer samples can beformed or deposited. The substrate can be of any suitable material, andpreferably consists essentially of materials that are inert with respectto the polymer samples of interest. Certain materials will, therefore,be less desirably employed as a substrate material for certainpolymerization reaction process conditions (e.g., hightemperature—especially temperatures greater than about 100° C. or highpressures) and/or for certain reaction mechanisms. Stainless steel,silicon, including polycrystalline silicon, single-crystal silicon,sputtered silicon, and silica (SiO₂) in any of its forms (quartz, glass,etc.) are preferred substrate materials. Other known materials (e.g.,silicon nitride, silicon carbide, metal oxides (e.g., alumina), mixedmetal oxides, metal halides (e.g., magnesium chloride), minerals,zeolites, and ceramics) may also be suitable for a substrate material insome applications. Organic and inorganic polymers may also be suitablyemployed in some applications of the invention. Exemplary polymericmaterials that can be suitable as a substrate material in particularapplications include polyimides such as Kapton™, polypropylene,polytetrafluoroethylene (PTFE) and/or polyether etherketone (PEEK),among others. The substrate material is also preferably selected forsuitability in connection with known fabrication techniques. As to form,the sample containers formed in, at or on a substrate can be preferably,but are not necessarily, arranged in a substantially flat, substantiallyplanar surface of the substrate. The sample containers can be formed ina surface of the substrate as dimples, wells, raised regions, trenches,or the like. Non-conventional substrate-based sample containers, such asrelatively flat surfaces having surface-modified regions (e.g.,selectively wettable regions) can also be employed. The overall sizeand/or shape of the substrate is not limiting to the invention. The sizeand shape can be chosen, however, to be compatible with commercialavailability, existing fabrication techniques, and/or with known orlater-developed automation techniques, including automated sampling andautomated substrate-handling devices. The substrate is also preferablysized to be portable by humans. The substrate can be thermallyinsulated, particularly for high-temperature and/or low-temperatureapplications. In preferred embodiments, the substrate is designed suchthat the individually addressable regions of the substrate can act aspolymerization reaction vessels for preparing a polymerization productmixture (as well as sample containers for the two or more differentpolymer samples during subsequent characterization thereof. Glass-lined,96-well, 384-well and 1536-well microtiter-type plates, fabricated fromstainless steel and/or aluminum, are preferred substrates for a libraryof polymer samples. The choice of an appropriate specific substratematerial and/or form for certain applications will be apparent to thoseof skill in the art in view of the guidance provided herein.

[0098] The library of polymer materials can be a combinatorial libraryof reaction product mixtures such as polymerization product mixtures.Polymer libraries can comprise, for example, polymerization productmixtures resulting from polymerization reactions that are varied withrespect to, for example, reactant materials (e.g., monomers,comonomers), catalysts, catalyst precursors, initiators, additives, therelative amounts of such components, reaction conditions (e.g.,temperature, pressure, reaction time) or any other factor affectingpolymerization. Design variables for polymerization reactions are wellknown in the art. See generally, Odian, Principles of Polymerization,3^(rd) Ed., John Wiley & Sons, Inc. (1991). A library of polymer samplesmay be prepared in arrays, in parallel polymerization reactors or in aserial fashion. Exemplary methods and apparatus for preparing polymerlibraries—based on combinatorial polymer synthesis approaches—aredisclosed in copending U.S. patent application Ser. No. 09/211,982 ofTurner et al. filed Dec. 14, 1998, copending U.S. patent applicationSer. No. 09/227,558 of Turner et al. filed Jan. 8, 1999, copending U.S.patent application Ser. No. 09/235,368 of Weinberg et al. filed Jan. 21,1999, and copending U.S. provisional patent application Ser. No.60/122,704 entitled “Controlled, Stable Free Radical Emulsion andWater-Based Polymerizations”, filed Mar. 9, 1999 by Klaemer et al. underAttorney Docket No. 99-4. See also, PCT Patent Application WO 96/11878.

[0099] The libraries can be advantageously characterized directly,without being isolated, from the reaction vessel in which the polymerwas synthesized. Thus, reagents, catalysts or initiators and otheradditives for making polymers may be included with the polymer samplefor characterization or screening.

[0100] While such methods are preferred for a combinatorial approach topolymer science research, they are to be considered exemplary andnon-limiting. As noted above, the particular polymer samplescharacterized according to the methods and with the apparatus disclosedherein can be from any source, including, but not limited topolymerization product mixtures resulting from combinatorially synthesisapproaches.

[0101] Mini- and Micro-Scale Applications

[0102] The methods of the present invention can be applied in connectionwith “normal” scale HPLC systems, and can also be applied to smallerscale systems—including particularly mini-scale systems and micro-scalesystems. As used herein, mini-scale systems are considered to includethose having mobile-phase supply conduits with a diameter ranging fromabout 3 mm to about 500 μm and micro-scale systems are considered toinclude those having mobile-phase supply conduits with a diameter ofabout 500 μm or less. Corresponding dimensions, in terms of hydraulicradius can be considered for other than circular cross-sectional areas.

[0103] The following examples further exemplify the invention. Theyshould be considered non-limiting.

EXAMPLE 1

[0104] The Chromatographic/Flow injection system was equipped with apump and autosampler (Alliance system 2690, Waters), and achromatographic column (or filter). The mobile phase coming out of theChromatographic/Flow injection system went through the mixer into thephotodiode array detector (Model 996, Waters), set up to measureabsorbance at 350 nm. Another pump (Model 515, Waters) for pumping aderivatization/precipitation agent from a prepared container to themixer was connected.

EXAMPLE 2

[0105] In the first experiment of this example, 10 □L of the solution ofthe narrow polyisobutylene standard (pib1100, Mw=1,110,000) from PolymerStandard Service was injected (volume) into the system with a metalfilter holder with a 0.5 □m pore size frit (Valco) connected instead ofseparation column. Tetrahydrofuran at 1 mL/min was used as a mobilephase. No derivatization/precipitation agent was pumped into the mixerat this time. The data were acquired for 2 minutes. No significantresponse was seen under these conditions.

[0106] In the second experiment, the procedure was repeated as describedabove; however, methanol at 1 mL/min was mixed with the mobile phasecoming out of the Chromatographic/Flow injection system using the mixerinserted between the system and detector. Significantly large peak wasobserved on the chromatogram. In the third experiment, the procedure wasrepeated again using water at 0.2 mL/min as theprecipitation/derivatization agent instead of methanol. Here again,significantly large peak was observed on the chromatogram.

[0107] All three chromatographic traces are shown in FIG. 6. The exampleclearly demostrates that adding the precipitation/derivatization agentmakes previously invisible polymers visible. The wavelength from thevisual light range has been chosen to demostrate ability to detectpolymer in a very efficient way using optical detection system that mayuse even visual light. Such a detector can be very easy and cheap tobuild since it wouldn't require any complicated light sources andwavelength filtering.

EXAMPLE 3

[0108] In this example, water at 0.1 mL/min was used as theprecipitation/derivatization agent and all the other experimentalconditions were the same as those described in the

Example 2

[0109] First, three narrow polyisobutylene standards differing inmolecular weights (Mw 1,1M; Mw 355 k; and 10 k, Polymer StandardService) were injected subsequently into the system. All three polymersgave a significant detector response as compared to the blank experimentwith no injection (FIG. 7A).

[0110] Then, three narrow polystyrene standards differing in molecularweights (Mw 3M; Mw 215 k; and 11 k, Polymer Laboratories) were injectedsubsequently into the system. No detector response was observed afterinjecting these standards (FIG. 7B). This example demonstrates thatunder certain conditions the detection can be group selective and allowus to distinguish one group of polymers from the others based on thedifferences in the chemical compositions.

EXAMPLE 4

[0111] In this example, a separation column (PL-gel MiniMix-D, 250×4.6mm, 5 □m; Polymer Laboratories) was used instead of the filter, andwater at various flow rates was applied as theprecipitation/derivatization agent. The data were collected for 6 minper injection. All the other experimental conditions were the same asthose described in the Example 2.

[0112] In the first experiment, a mixture of three narrow polystyrenestandards differing in molecular weights (Mw 3M; Mw 70 k; and 11 k,Polymer Laboratories) was injected into the system having mobile phasecoming out of the Chromatographic/Flow injection system mixed with waterpumped into the mixer at 0.1 mL/min. No detector response was observedunder these conditions (FIG. 8D).

[0113] In the second experiment, the same mixture of polystyrenestandards was injected into the system with water at 0.15 mL/min used asthe precipitation/derivatization agent. Only a peak of the polystyrenestandard having the highest molecular weight (3M) was observed (FIG.8C).

[0114] In the third experiment, the same mixture of standards wasinjected into the system with water at 0.3 mL/min used as theprecipitation/derivatization agent. Two peaks corresponding to the twopolystyrene standard having molecular weight of 3M and 70 k appeared onthe chromatogram (FIG. 8B).

[0115] In the fourth experiment, the same mixture of standards wasinjected into the system with water at 0.5 mL/min used as theprecipitation/derivatization agent. Three peaks corresponding to all ofthe injected polystyrene standard (3M, 70 k, 11 k) appeared on thechromatogram obtained under these conditions (FIG. 8A).

[0116] This example demonstrates that under certain conditions thedetection can be molecular weight selective and allow us to distinguishpolymers of the same chemical composition but of different molecularweights.

EXAMPLE 5

[0117] In this example, a separation column (PL-gel MiniMix-D, 250×4.6mm, 5 □m; Polymer Laboratories) was used instead of the filter, andeither no precipitation/derivatization agent or water at 0.4 mL/min wasapplied to influence the detection. UV absorbance (photodiode arraydetector, Model 996, Waters, set up to absorbance detection at 254 nm)and Light-Scattering at 900 (MiniDawn, Wyatt Technologies) detectorswere used in a series for the following experiments. The data werecollected for 5 min per injection. All tie other experimental conditionswere the same as those described in the Example 2.

[0118] First, the narrow polystyrene standard of 214 k was injected intothe system with no precipitation/derivatization agent. A response wasobserved on both UV detectors. Then, the injection was repeated into thesystem with water at 0.4 mL/min applied as precipitation/derivatizationagent. Much stronger response was observed on both UV (FIG. 9A) and LS(FIG. 9B) detectors.

[0119] This demonstrates that such a post column treatment can greatlyenhance the detection of polymers using various optical detectionsystems. This may have a vast applicability for detecting a trace amountof polymers which would be otherwise far below the sensitivity limit ofa detector. Also, changing the derivatization/precipitation conditionsallow to tune up the sensitivity of the detection in order to fit alarge variety of concentrations within in a diverse library of polymersamples.

[0120] In light of the detailed description of the invention and theexamples presented above, it can be appreciated that the several objectsof the invention are achieved. The explanations and illustrationspresented herein are intended to acquaint others skilled in the art withthe invention, its principles, and its practical application. Thoseskilled in the art may adapt and apply the invention in its numerousforms, as may be best suited to the requirements of a particular use.Accordingly, the specific embodiments of the present invention as setforth are not intended as being exhaustive or limiting of the invention.

I claim:
 1. A method for characterizing a plurality of samples with aliquid chromatography system, the method comprising supplying a mobilephase in parallel through each of first and second chromatographiccolumns of a liquid chromatography system, injecting first and secondsamples into the mobile phase of the first and second chromatographiccolumns, respectively, separating at least one sample component of theinjected first and second samples from other sample components thereofin the respective chromatographic columns, treating the at least oneseparated sample component of the first and second samples to change aproperty of at least one separated sample component thereof, anddetecting a property of the treated sample component of the first andsecond samples.
 2. The method of claim 1 wherein the at least oneseparated sample component of the first and second samples are treatedto change an optical property thereof, and the detected property of thetreated sample component is an optical property.
 3. The method of claim1 wherein treating comprises precipitating the at least one separatedsample component.
 4. The method of claim 1 wherein treating comprisesselectively precipitating the at least one separated sample component.5. The method of claim 1 wherein the samples are polymer samples, andtreating comprises precipitating the at least one separated samplecomponent by combining the chromatographic eluant with a non-solvent forthe at least one separated sample component.
 6. The method of claim 1wherein the samples are polymer samples, and treating comprisesprecipitating the at least one seperated sample component by controllingthe temperature of the chromatographic eluant.
 7. The method of claim 1wherein treating comprises derivatizing the at least one separatedsample component.
 8. The method of claim 1 wherein treating comprisesselectively derivatizing the at least one separated sample component. 9.The method of claim 1 wherein at least two or more sample components ofthe injected first and second samples are separated from each other andfrom other components thereof.
 10. The method of claim 1 wherein the atleast one separated sample component of the first and second samples istreated to be selectively detectable over one or more other samplecomponents thereof, and a property of the treated sample is selectivelydetected thereover.
 11. The method of claim 1 wherein ten or moresamples are injected into the mobile phase of the first and secondchromatographic columns.
 12. The method of claim 1 wherein forty or moresamples are injected into the mobile phase of the first and secondchromatographic columns.
 13. The method of claim 1 wherein eighty ormore samples are injected into the mobile phase of the first and secondchromatographic columns.
 14. The method of claim 1 wherein 96*N samplesare injected into the mobile phase of the first and secondchromatographic columns, where N is an integer ranging from 1 to
 5. 15.The method of claim 1 wherein ten or more different samples are injectedinto the mobile phase of the first and second chromatographic column.16. The method of claim 1 wherein the samples are polymer samples. 17.The method of claim 1 wherein a property of at least one of theseparated sample components of the first and second samples is detectedin series.
 18. The method of claim 1 wherein a property of at least oneof the separated sample components of the first and second samples isdetected in parallel.
 19. The method of claim 1 wherein a property of atleast one of the separated sample components of the first and secondsamples is detected with an optical detector. The method of claim 1further comprising determining a property of interest.
 20. A method forcharacterizing a plurality of polymer samples with a liquidchromatography system, the method comprising supplying a mobile phase inparallel through each of first and second chromatographic columns of aliquid chromatography system, injecting first and second polymer samplesinto the mobile phase of the first and second chromatographic columns,respectively, separating at least one sample component of the injectedfirst and second polymer samples from other sample components thereof inthe respective chromatographic columns, precipitating or derivatizing atleast one separated sample component of the first and second polymersamples, and detecting an optical property of a precipitated orderivatized sample component of the first and second polymer samples.21. A method for characterizing a plurality of polymer samples with aliquid chromatography system, the method comprising supplying a mobilephase in parallel through each of two or more chromatographic columns ofa liquid chromatography system, the mobile phase comprising a solventfor ten or more polymer samples of interest, injecting the ten or moredifferent polymer samples of interest into the mobile phase of the twoor more chromatographic columns, separating at least one samplecomponent of the injected ten or more polymer samples from other samplecomponents thereof in the respective chromatographic columns, combiningthe eluant of the chromatographic column with a non-solvent for the tenor more polymer samples of interest to precipitate the at least oneseparated sample component thereof, and detecting an optical property ofthe precipitated sample component of the ten or more polymer samples.22. A method for characterizing a plurality of samples with a liquidchromatography system, the method comprising supplying a mobile phase inparallel through each of first and second chromatographic columns of aliquid chromatography system, injecting first and second samples intothe mobile phase of the first and second chromatographic columns,respectively, separating at least one sample component of the injectedfirst and second samples from one or more other sample componentsthereof in the respective chromatographic columns, controlling thecomposition or the temperature of the mobile-phase after chromatographicseparation, such that an optical property of one or more of theseparated sample components of the first and second samples isselectively changed relative to the optical properties of other samplecomponents of the first and second samples, and selectively detectingthe changed optical property of the at least one separated samplecomponent of the first and second samples.
 23. A method forcharacterizing a plurality of samples with a flow-injection analysissystem, the method comprising supplying a mobile phase in parallelthrough each of first and second flow detectors of a flow-injectionanalysis system, injecting first and second polymer samples into themobile phase of the first and second flow detectors, respectively,controlling the composition, the flow-rate or the temperature of themobile-phase after injection of the first and second polymer samples,such that an optical property of one or more of the sample components ofthe first and second polymer samples is selectively changed relative tothe optical properties of other sample components of the first andsecond polymer samples, and selectively detecting the changed opticalproperty of the at least one sample component of the first and secondpolymer samples.